Translation of abstract (English)

Fibroblast growth factor 2, a mediator of tumor-associated angiogenesis, is a mitogenic growth factor involved in various cellular processes. It is released by an unconventional secretory pathway independent of the ER/Golgi system. Due to its strong biomedical relevance it is of great interest to elucidate the molecular machinery involved in non-classical export. To analyze unconventional secretion, different model systems were established during this study including a FACS-based system which allows for a quantitative analysis of exported material bound to the cell surface and an analysis system employing confocal microscopy to analyze non-classical export qualitatively employing specific antibodies. Additionally, various biochemical analysis methods employing immobilized antibodies, as well as labelling of cell surface proteins using a membrane-impermeable biotinylation reagent to quantify exported FGF2 reporter molecules were established. In the second part of this thesis, these systems were used to analyze the folding state of FGF2 during unconventional secretion. The first experimental approach, termed DHFR fusion protein system, prevents unfolding during membrane translocation by aminopterin-dependent stabilization of a DHFR domain fused to FGF2. It could be shown that export of FGF2 is not affected under conditions where protein unfolding is prevented, although, based on the same system, mitochondrial import could be blocked. These findings suggest that export of FGF2 does not require unfolding. The second strategy, termed piggyback export analysis system, monitors the folding state of FGF2 and investigates potential means of quality control associated with unconventional secretion. The system is based on the export of non-covalent cytosolic complexes formed between two interacting domains, one fused to FGF2 and the other to non-exported GFP. To this end, a certain degree of piggyback export, could be detected, however, the efficiency was found to be low. In any case, the results are consistent with those obtained with the DHFR system in that it appears likely that FGF2 remains folded during membrane translocation. These findings are also supported by recent observations made in our laboratory pointing to a role of heparan sulfate proteoglycans as export receptors requiring FGF2 to be folded during export.